Second harmonic generation (SHG) is a nonlinear optical process which may be configured as a surface-selective detection technique that enables detection of conformational change in proteins and other biological targets (as described previously, for example, in U.S. Pat. Nos. 6,953,694 and 8,497,703). In order to deploy SHG-based detection of conformational change in a convenient and high throughput format, it will be advantageous to design novel methods for immobilizing proteins on substrate surfaces, and novel mechanisms for rapid, precise, and interchangeable positioning of substrates (comprising the tethered biological targets to be analyzed) with respect to the optical system used to deliver excitation light, which ensure that both a high degree of orientation of the protein molecules at the optical interface and efficient optical coupling between the excitation light and the substrate surface are maintained.
The methods and compositions disclosed herein provide means for tethering or immobilizing protein molecules on optical interfaces in a manner that ensures both uniform surface coverage of the optical interface and a high degree of orientation of the molecules, thereby providing for significant enhancements of SHG and other nonlinear optical signals and improved signal-to-noise ratios.